Project Summary Antimicrobial resistance is a global concern. While bacterial resistance is becoming more widespread, antibiotic development has shrunk significantly over the past 25 years. This emphasizes a critical need for the development of new antibiotics. Both Gram-negative and Gram-positive bacteria are surrounded by cell walls made of peptidoglycan that protect the cells against osmotic pressure. Peptidoglycan biosynthesis is a well- established target for antibiotic development. MraY (phospho-MurNAc-pentapeptide translocase) is an essential membrane protein that catalyzes the first membrane step of bacterial cell wall biosynthesis. MraY is the target of many natural product antibiotics, making it a promising target for antibiotic development. MraY belongs to a subfamily of the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) superfamily. The PNPT superfamily includes enzymes responsible for cell envelope polymer synthesis in bacteria and N-linked glycosylation in eukaryotes. Despite the therapeutic potential of MraY and physiological importance of the PNPT superfamily in general, a mechanistic understanding of the enzyme and its superfamily has been elusive largely due a lack of detailed structural information. The goal of this proposal is to elucidate the mechanisms of MraY function and its inhibition by natural product antibiotics using structural and functional studies. We have chosen MraY from Aquifex aeolicus (MraYAA) for structural studies. MraYAA is an excellent model system to study given the high sequence conservation of its active site with MraYs from pathogenic bacteria. We recently solved the structure of MraYAA, the first structure of a member of the PNPT family. The structure not only provides mechanistic insights, but also raises many new questions. On the basis of our structural and functional studies of MraYAA, we will extend our functional studies to MraYs from pathogenic bacteria. Toward this end, we have managed to produce homogeneous and enzymatically active recombinant MraY from pathogenic bacteria. These new results have led us to propose the following four aims: 1) Understanding the structural basis of MraYAA catalysis; 2) Understanding the structural basis of MraYAA inhibition by natural nucleoside antibiotics; 3) Enzymatic and cell-based characterization of MraYAA function; 4) Biochemical and functional characterization of MraY from pathogenic bacteria. These studies will advance our understanding of the mechanism of MraY function and inhibition significantly, which will provide a platform for the future development of novel antibiotics.